Caveolae and L-type Calcium Channels in Cardiomyocytes

项目概述：钙通过心肌细胞表面膜上的l型钙通道内流调节多种细胞过程，包括收缩、分泌、细胞信号传导和基因表达。本研究的长期目标是：1)基于相关蛋白的独特大分子复合物来区分心肌细胞中l型钙通道的不同群体；2)确定这些不同的通道群如何特异性地促进各种钙调节的细胞过程；3)了解不同人群钙通道的改变对心血管疾病的影响。这些目标将首先集中于Cav1.2 l型Ca通道，该通道定位于由特征蛋白caveolin-3 （caveolin-3）定义的称为caveolae的特殊膜微域。将定义Cav1.2通道在与最近发现的Cav-3突变相关的长QT综合征发生中的作用。利用免疫沉淀技术、GST-Cav-3下拉、免疫共聚焦显微镜、免疫金电镜、靶向蛋白siRNA敲除、Cav1.2通道和关键相关蛋白的异种表达以及小鼠cav3的条件敲除，我们将解决三个具体目标：1)确定心室肌细胞中定位于小窝的Cav1.2通道的相对丰度和组成；2)确定Cav-3及其相关核心支架蛋白是否对心室肌细胞正常基础L型Ca电流及其（¿- ar）调控至关重要；3)确定长qt相关的Cav-3基因突变对Cav1.2 L型Ca通道的影响。

DESCRIPTION (provided by applicant): Project Summary: The influx of Ca through L-type Ca channels in surface membrane of cardiomyocytes regulates a variety of cellular processes including contraction, secretion, cell signaling, and gene expression. The long-term goals of this research are 1) to discriminate between distinct populations of L-type Ca channels in cardiomyocytes based on the unique macromolecular complex of associated proteins; 2) to determine how these different populations of channels specifically contribute to various Ca-regulated cellular processes; and 3) to understand how alterations in the different populations of Ca channels contribute to cardiovascular disease. These goals will be approached initially focusing on Cav1.2 L-type Ca channels localized to the specialized membrane microdomains known as caveolae which are defined by the signature protein caveolin-3 (Cav-3). The contribution of Cav1.2 channels to the genesis of the long QT syndrome associated with recently identified mutations in Cav-3 will be defined. Using immunoprecipitation techniques, GST-Cav-3 pull-down, immunoconfocal microscopy, immunogold electron microscopy, siRNA knockdown of targeted proteins, heterologous expression of Cav1.2 channels and key associate proteins, and conditional knockout of Cav-3 in mice, we will address three specific aims: 1) Define the relative abundance and composition of the Cav1.2 channels localized to caveolae in ventricular myocytes; 2) Determine if Cav-3 and associated core scaffolding proteins are essential for normal basal L-type Ca current and its (¿-AR regulation in ventricular myocytes; 3) Determine the impact of long QT-related Cav-3 genetic mutations on Cav1.2 L- type Ca channels. Relevance: These studies will provide molecular definition of the subpopulation of Ca channel proteins localized to caveolae and thus offer mechanistic insights into the genesis of the long QT syndrome and life-threatening arrhythmias associated with mutations in the signature protein of caveolae in the heart, caveolin-3. Furthermore, this research will impact our understanding of heart failure and atrial fibrillation given that alterations in caveolae and Ca channels have been detected in these diseases. Ultimately, a molecular understanding of caveolae and L-type Ca2+ channels has the potential to inspire new therapies for these prevalent cardiovascular diseases.